**2.5. Hyaluronic acid (HA)-modified liposomes**

To develop an effective VADS, Noormehr et al*.* fabricated 500-nm-sized PLGA NPs which were covalently conjugated with recombinant Ags Leishmanial CPA (cysteine peptidase A) and CPB, and proved that mice intra-peritoneally immunized with the inhomogeneous Ag-NPs secreted high levels of NO (nitric oxide) by peritoneal MPs and high levels of IFN-γ by splenocytes, which significantly lowered *Leishmania major* burden, suggesting the Ag-conjugated PLGA NPs can be used as a VADS able to deliver vaccines to protect against the tough pathogen of parasites [54]. To investigate the function of multiple adjuvant-combined VADS, Ebrahimian and colleagues formulated the TLR 7/8a resiquimod- or TLR4a MPLA-loaded PLGA NPs which were physically covered with polyethylenimine (PEI) forming PLGA/PEI NPs and then mixed with CpG ODN (cytosine-phosphorothioate-guanine oligodeoxynucleotide) to engender a complexed entity of resiquimod- or MPLA-PLGA NPs/PEI-CpG ODN [55]. Given to BALB/c mice, the multiple adjuvant-constituted PLGA NPs loaded with Ags induced robust and efficient immune responses, as confirmed by evaluation of vivo cytokine (IFN-γ, IL-4, and IL-1β) secretion and antibody (IgG1 and IgG2a) production, demonstrating using a combination of adjuvants in a context-dependent manner may a feasible strategy for engineering a potent PLGA-based VADS. To make subunit vaccines suitable for immunization via skin, which is an attractive but also very challenging immunization site due to the presence of affluent APCs while difficulty of administration, recently, Bouwstra's group fabricated the hyaluronan (HA)-based dissolving microneedles (MNs) entrapped with PLGA NPs which co-encapsulated ovalbumin (OVA) as an Ag and poly(I:C) as an adjuvant for intradermal immunization [56]. Further investigation indicated that the immunogenicity of the PLGA NPs after administration of dissolving MNs was compared with that of hollow MN-delivered PLGA NPs in mice, while immunization with free Ag in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by PLGA NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN, suggesting several critical parameters should be fully evaluated in engineering the PLGA NP-loaded dissolving MNs as an intradermal

36 Immunization - Vaccine Adjuvant Delivery System and Strategies

VADS to avoid unnecessary efforts on the complexed formulations.

At present, still a large fraction of vaccines require a multiple dosing schedule with a 1- to 2-month gap between administrations to guarantee establishing the Ag-specific immunity strong enough to protect recipients, as such, however, engendering a big challenge to worldwide vaccination, especially, in the developing countries, where healthcare workers are not only in shortage but also confronting difficulty in reaching the subjects multiple times to administer booster shots [57]. Conceptually, this challenge may be conquered using a VADS that are constructed with a functional carrier which release vaccine ingredients in pulses with an appropriate time gap between vaccinations, thus simplifying the vaccination schedule to consist of only once injection to exclude additional visits by a healthcare worker. For this, Tzeng et al*.* engineered a controlled release VADS consisting of bPEI (branched PEI)-modified PLGA microparticles which contained in inner core Ags of IPV (inactivated polio vaccine with three antigens) and an Ag stabilizer poly(L-lysine) [58]. Further investigation indicated that the bPEI-PLGA microparticles stabilized IPV in its active conformation inside the particles for months but in an aqueous medium released two bursts of IPV with an interval of just 1 month, vividly mimicking a typical twice vaccination schedule. Moreover, one injection of the controlledrelease formulations elicited a similar or better Ag neutralizing response in rats compared to Hyaluronic acid (HA) as a polysaccharide consists of alternating units of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (Glc-NAc), connected to each other with β-1,3- and β-1,4 glycosidic bonds, having nearly perfect chemical repeats except for occasional deacetylated glucosamine residues to form a very hydrophilic linear high molecular weight (HMW) biopolymer [59]. HA ranges in size from 5 kDa to 10 million Da (corresponding to 25,000 disaccharide units), with the most common forms of 1–8 million Da in humans and can absorb water to expand its solid volume by up to 1000 times forming a very viscous and elastic gel [60]. HA GlcA carboxyl group is dissociated at physiological pH values to engender a negatively charged polymer which is readily combining with the most prevalent extracellular cation of Na<sup>+</sup> to form sodium hyaluronate, suggesting that the molecule is not ionized [59]. In fact, while native HA with a high molecular weight (HMW) acts mainly as a constructive stuff and a control on tissue hydrodynamics, low molecular weight (LMW) HA usually participates in cell signaling through interaction with certain types of cell surface receptors, such as CD44 as the primary one, but also TLR2 and TLR4, thus contributing to several physiological and biological activities [61, 62].

As an abundant endogenous polymer, HA has been widely exploited to construct the functional carriers for delivering various bioactives with expectation of improving human health, given many of its desirable merits that can be employed for optimizing delivery effects [63]. Most attractively, HMW HA possesses numerous physicochemical and physiological features, such as biocompatibility, biodegradation, mucoadhesive property, bearing negative charges in a neutral condition, possessing active groups allowing various functional modifications that are all beneficial characters for engineering carriers to deliver agents [64]. Another interesting aspect lies in that LMW HA binds to several receptors, especially CD44, which is ubiquitously expressed on various cells, and especially overexpressed on many types of cancer cells, presenting bases for developing a tumor-targeting drug delivery system (DDS) with attractive advantages, such as the ease of associating drugs with the polysaccharide or its carrier thus solving any solubility problems, improving a drug's blood plasma half-life thus playing a similar role to PEG, and high tumor-targeting efficiency, and as such is currently the main trend in the HA-based delivery research [65]. More recently, LMW HA is focused on its ability to activate CD44 or TLRs on immune cells involving regulation of certain signaling pathways associated with APC maturation, cytokine production and innate immune responses for immunization, and even, in a CD44- and TLR4-independent manner, to enhance CCR7 expression on DCs promoting DC recruitment to tumor regional lymph nodes and restraining DC migration toward tumor tissue [62, 66–68], thus providing not only a comprehensive option for engineering functional nanoparticles fitting a VADS, but also a promising candidate for improving DC maturation in the context of DC-based vaccine development.

Up to now, most of the HA-based carriers used as a VADS have been developed by making use of the "nonbioactive" properties, which has little physiological interference on the body and as such, is used just as constructive stuff. Moon's group formulated the HA-PEG-shelled cationic DOTAP/DOPE liposomes carrying F1-V, a candidate recombinant antigen for *Yersinia pestis*, as a stable and potent nasal VADS, which exhibited markedly decreased cytotoxicity associated with DOTAP liposomes to BMDCs, and when further incorporated with MPLA promoted BMDC maturation and induced a strong Th1/Th2-balanced immunoresponse toward Ags, as evidenced by high titers of F1-V-specific total IgG, IgG1, and IgG2c produced in intranasally immunized mice [69]. Huang's group engineered mLCP (the mannosylated lipid-calcium-phosphate NPs) and LPHa NPs (liposome-protamine-HA-anisamide NPs) for, respectively, targeting delivery of the tumor antigen Trp 2 peptide/CpG ODN to APCs and the TGF-β-silencing siRNA to tumor cells which overexpress sigma receptors with a ligand of anisamide [70]. They demonstrated that the delivery of Trp 2/CpG ODN to DCs by mLCPbased VADS elicited a potent systemic immune response to tumors in mice but generated, to later stage B16F10 melanoma, a marginal efficacy, which however, was remarkably boosted through silencing the immune-suppressive cytokine TGF-β in tumor cells with siRNA-loaded LPHa NPs to engender increased tumor infiltrating CD8+ T cells and decreased regulatory T cells within tumor microenvironment. Wu's group fabricated a microneedle array (MA) with HA with a deep cave formed in the basal portion of each microneedle, into which BCG (Bacille Calmette-Guerin bacillus) powder could be packaged directly, thus producing a painless VADS of MA-BCG, which after vaccination by patching on skin of mice caused no overt skin irritation, but elicited strong humoral and cellular immunity comparable to that of intradermal immunization [71]. Notably, other researchers showed in a clinical trial that HA-constructed MA containing trivalent influenza hemagglutinins (A/California/07/2009 (H1N1), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008, 15 μg each) induced immune responses against A/H1N1 and A/H3N2 strains equal to that by subcutaneous injection groups without stirring severe local or systemic adverse reactions and engendered the efficacy against the B strain much stronger than that by the injection group, proving HA-MA a promising practical use as an easy and effective method to replace conventional injection systems [72].

against the hepatitis B antigen in the cationic NP group, indicating that the protamine/HA NPs depending on physical features may be an effective VADS for delivering subunit HBV vaccines [74]. Kim et al*.* using LMW HA (215 kDa) synthesized HA-OVA conjugates, which proved able to facilitate DC maturation *in vitro* and, after topical application to penetrate into the dermis in murine skins, efficiently induced secretion of the anti-OVA IgG levels in serum as well as IgA levels in bronchioalveolar lavage, which could promptly respond to an OVA challenge after 8 weeks rendering a strong immune-recall humoral response, especially, under the condition of pretreatment of the skin using nonablative fractional laser beams to save Ag dose, strongly supporting of the adjuvant role that LMW HA can play for developing

Polymeric Nanoparticles Engineered as a Vaccine Adjuvant-Delivery System

http://dx.doi.org/10.5772/intechopen.81084

39

In summary, HA is a biodegradable and safe endogenous polymer, which can be used to engineer either inert NPs with high molecular weight HA or cell-targeting NPs with low molecular weight HA based on the fact its selective binding to several receptors, such as CD44 and TLR4, which may possibly trigger innate immune responses, allowing HA-based NPs to be conveniently employed to construct multifunctional VADS able to efficiently deliver

At present, various polymeric NP-based VADSs have been designed for delivering as well as adjuvanting vaccines to elicit robust Ag-specific humoral and cellular immunity at both systemic and mucosal levels to provide extensive protection against infectious pathogens. In particular, many types of polymeric NPs can be tailored as a multiple functional VADS to render Ags lysosome escape after APC uptake, allowing vaccine epitopes not only to avoid being degraded into null pieces but also to selectively bind to MHC-I or -II for presentation to dictate immune responses toward a Th1 and/or Th2 pathway to set up immunity fitting medical aims. Encouragingly, a few of the polymeric NP VADS-based subunit vaccines have been approved, as mile stones, for clinical vaccination, typical products including the virosomebased hepatitis A vaccine (Epaxal®) and influenza vaccine (Inflexal V®), VLP-based HBV vaccine and malaria vaccine. Undoubtedly, as many of the uncertainties and problematic issues associated with polymeric NPs, such as safety of synthetic materials, scale-up production, and cost of products, are ultimately resolved, more and more polymeric NP VADS-based

This work was financially supported by the National Natural Science Foundation of China (Grant number 81703449), and partially by Department of Science & Technology of Anhui Province for Natural Science Research Project (Grant number 1708085QH195), and also by the Department of Education of Anhui Province (Grant numbers KJ2016SD28, gxfxZD2016045).

vaccines will be developed and licensed to enter markets.

the painless topical VADS [75].

various subunit vaccines.

**Acknowledgements**

**3. Conclusions**

Recently, Hahn's group conjugated an antigenic peptide of myostatin fragment (MstnF) to HA with a LMW (17 kDa) for transdermal vaccination against Duchenne muscular dystrophy (DMD), which is a neuromuscular disorder accompanied with muscle weakness and wasting with myostatin emerging as a key negative regulator [73]. In vivo experiments demonstrated that HA-MstnF conjugates efficiently penetrated into deep skin layers, and HA exerted a boosting effect on the immunization of MstnF in the transdermally vaccinated mice, which not only secreted high levels of antibody titers against myostatin but also showed a significant improvement in the pathological status of skeletal musculature as well as functional behaviors. Gonzalez-Aramundiz et al. prepared protamine/LMW HA (162 kDa) NPs using a mild ionic cross-linking technique and showed that in vitro Ag (rHBsAg)-loaded anionic NPs (protamine/HA of 1:4, w/w) induced the secretion of cytokines including TNFα, IL-1α, and IL-6 by macrophages more efficiently than the cationic NPs (protamine/HA of 4:1), whereas in mice, by either intramuscular or intranasal administration, the cationic NPs induced more robust immune responses than the anionic NPs did, as proved by the higher levels of the IgG against the hepatitis B antigen in the cationic NP group, indicating that the protamine/HA NPs depending on physical features may be an effective VADS for delivering subunit HBV vaccines [74]. Kim et al*.* using LMW HA (215 kDa) synthesized HA-OVA conjugates, which proved able to facilitate DC maturation *in vitro* and, after topical application to penetrate into the dermis in murine skins, efficiently induced secretion of the anti-OVA IgG levels in serum as well as IgA levels in bronchioalveolar lavage, which could promptly respond to an OVA challenge after 8 weeks rendering a strong immune-recall humoral response, especially, under the condition of pretreatment of the skin using nonablative fractional laser beams to save Ag dose, strongly supporting of the adjuvant role that LMW HA can play for developing the painless topical VADS [75].

In summary, HA is a biodegradable and safe endogenous polymer, which can be used to engineer either inert NPs with high molecular weight HA or cell-targeting NPs with low molecular weight HA based on the fact its selective binding to several receptors, such as CD44 and TLR4, which may possibly trigger innate immune responses, allowing HA-based NPs to be conveniently employed to construct multifunctional VADS able to efficiently deliver various subunit vaccines.
